LOCUS Exported 9223 bp ds-DNA circular SYN 23-JUL-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 9223) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Jul 31, 2019 from SnapGene Viewer 4.3.8 https://www.snapgene.com FEATURES Location/Qualifiers source 1..9223 /organism="recombinant plasmid" /mol_type="other DNA" promoter 62..302 /label=U6 promoter primer_bind 107..131 /label=pORANGE sequencing primer FW misc_feature 311..331 /label=Target sequence misc_RNA 332..407 /label=gRNA scaffold misc_feature 408..418 /label=Termination sequence misc_feature 509..511 /label=PAM misc_feature 512..531 /label=Target sequence CDS 541..1218 /codon_start=1 /label=mEos3.2 /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD NARR" CDS 1219..1224 /codon_start=1 /label=Linker /translation="AR" misc_feature 1226..1228 /label=PAM misc_feature 1229..1248 /label=Target sequence enhancer 1288..1573 /label=CMV enhancer primer_bind complement(1296..1315) /label=pORANGE sequencing primer RV promoter 1575..1852 /label=chicken beta-actin promoter intron 1853..2080 /label=hybrid intron CDS 2098..2100 /codon_start=1 /label=START /translation="M" CDS 2101..2127 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 2131..2151 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 2161..6261 /codon_start=1 /label=Cas9 /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6265..6285 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" polyA_signal 6331..6538 /label=bGH poly(A) signal repeat_region 6547..6687 /label=AAV2 ITR rep_origin 6762..7217 /direction=RIGHT /label=f1 ori /note="f1 stays for filamentous phages. It initiates a ""rolling circle"" replication in phage-infected bacteria and is useful if there is a need to produce vector as a single-stranded DNA" promoter 7499..7603 /label=AmpR promoter CDS 7604..8464 /codon_start=1 /label=AmpR /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8635..9223 /direction=RIGHT /label=ori /note="Ori for replication in E.coli" ORIGIN 1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 301 acgaaacacc gctgcctgcc cagtgccagg agttttagag ctagaaatag caagttaaaa 361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag 421 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc 481 agacaaatgg ctctagaagc ttgctagacc ctcctggcac tgggcaggca ggctagcagg 541 atgagtgcga ttaagccaga catgaagatc aaactccgta tggaaggcaa cgtaaacggg 601 caccactttg tgatcgacgg agatggtaca ggcaagcctt ttgagggaaa acagagtatg 661 gatcttgaag tcaaagaggg cggacctctg ccttttgcct ttgatatcct gaccactgca 721 ttccattacg gcaacagggt attcgccaaa tatccagaca acatacaaga ctattttaag 781 cagtcgtttc ctaaggggta ttcgtgggaa cgaagcttga ctttcgaaga cgggggcatt 841 tgcaacgcca gaaacgacat aacaatggaa ggggacactt tctataataa agttcgattt 901 tatggtacca actttcccgc caatggtcca gttatgcaga agaagacgct gaaatgggag 961 ccctccactg agaaaatgta tgtgcgtgat ggagtgctga cgggtgatat tgagatggct 1021 ttgttgcttg aaggaaatgc ccattaccga tgtgacttca gaactactta caaagctaag 1081 gagaagggtg tcaagttacc aggcgcccac tttgtggacc actgcattga gattttaagc 1141 catgacaaag attacaacaa ggttaagctg tatgagcatg ctgttgctca ttctggattg 1201 cctgacaatg ccagacgagc tcgagccctc ctggcactgg gcaggcaggg atcctaggac 1261 gcgtcctagg acgcgtccag aggtacccgt tacataactt acggtaaatg gcccgcctgg 1321 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca 1381 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 1441 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg 1501 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 1561 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 1621 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 1681 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 1741 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 1801 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 1861 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 1921 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 1981 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt 2041 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg 2101 tacccatacg atgttccaga ttacgcttcg ccgaagaaaa agcgcaaggt cgaagcgtcc 2161 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 2221 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 2281 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 2341 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 2401 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 2461 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 2521 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 2581 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 2641 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 2701 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 2761 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 2821 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 2881 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 2941 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 3001 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 3061 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 3121 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 3181 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 3241 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 3301 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 3361 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 3421 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 3481 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 3541 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 3601 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 3661 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 3721 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 3781 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 3841 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 3901 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 3961 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 4021 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 4081 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 4141 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 4201 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 4261 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 4321 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 4381 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 4441 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 4501 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 4561 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 4621 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 4681 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 4741 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 4801 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 4861 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 4921 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 4981 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 5041 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 5101 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 5161 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 5221 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 5281 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 5341 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 5401 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 5461 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 5521 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 5581 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 5641 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 5701 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 5761 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 5821 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 5881 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 5941 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 6001 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 6061 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 6121 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 6181 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 6241 ctgtctcagc tgggaggcga cagccccaag aagaagagaa aggtggaggc cagctaagaa 6301 ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 6361 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 6421 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 6481 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc 6541 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 6601 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 6661 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 6721 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 6781 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt 6841 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 6901 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 6961 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 7021 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 7081 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc 7141 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 7201 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 7261 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 7321 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 7381 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 7441 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 7501 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 7561 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 7621 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 7681 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 7741 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 7801 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 7861 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 7921 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 7981 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 8041 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 8101 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 8161 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 8221 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 8281 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc 8341 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 8401 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 8461 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 8521 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 8581 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 8641 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 8701 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 8761 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 8821 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 8881 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 8941 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 9001 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 9061 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 9121 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 9181 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaa //